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Doxorubicin (Dox) is an anthracycline chemotherapeutic agent used to treat breast, leukemia, and lymphoma malignancies. However, cardiotoxicity and inherent acquired resistance are major drawbacks, limiting its clinical application. We have previously shown that cyclic peptide [WR] 9 containing alternate tryptophan (W) and arginine (R) residues acts as an efficient molecular transporter. An amphiphilic cyclic peptide containing a lysine (K) residue and alternative W and R was conjugated through a free side chain amino group with Dox via a glutarate linker to afford [(WR) 8 WKβA]-Dox conjugate. Antiproliferative assays were performed in different cancer cell lines using the conjugate and the corresponding physical mixture of the peptide and Dox to evaluate the effectiveness of synthesized conjugate compared to the parent drug alone. [(WR) 8 WKβA]-Dox conjugate showed higher antiproliferative activity at 10 µM and 5 µM than Dox alone at 5 μM. The conjugate inhibited the cell viability of ovarian adenocarcinoma (SK-OV-3) by 59% and the triple-negative breast cancer cells MDA-MB-231 and MCF-7 by 71% and 77%, respectively, at a concentration of 5 μM after 72 h of incubation. In contrast, Dox inhibited the proliferation of SK-OV-3, MDA-MB-231, and MCF-7 by 35%, 63%, and 57%, respectively. Furthermore, [(WR) 8 WKβA]-Dox conjugate (5 µM) inhibited the cell viability of Dox-resistant cells (MES-SA/MX2) by 92%, while the viability of cells incubated with free Dox was only 15% at 5 μM. Confocal microscopy images confirmed the ability of both Dox conjugate and the physical mixture of the peptide with the drug to deliver Dox through an endocytosis-independent pathway, as the uptake was not inhibited in the presence of endocytosis inhibitors. The stability of Dox conjugate was observed at different time intervals using analytical HPLC when the conjugate was incubated with 25% human serum. Half-life (t 1/2 ) for [(WR) 8 WKβA]-Dox conjugate was (∼6 h), and more than 80% of the conjugate was degraded at 12 h. The release of free Dox was assessed intracellularly using the CCRF-CEM cell line. The experiment demonstrated that approximately 100% of free Dox was released from the conjugate intracellularly within 72 h. These data confirm the ability of the cyclic cell-penetrating peptide containing tryptophan and arginine residues as an efficient tool for delivery of Dox and for overcoming resistance to it.

Doxorubicin (Dox) is used for breast cancer, leukemia, and lymphoma treatment as an effective chemotherapeutic agent. However, Dox use is restricted due to inherent and acquired resistance and an 8-fold increase in the risk of potentially fatal cardiotoxicity. Hybrid cyclic-linear peptide [R 5 K]W 7 A and linear peptide R 5 KW 7 A were conjugated with Dox through a glutarate linker to afford [R 5 K]W 7 A-Dox and R 5 KW 7 A-Dox conjugates to generate Dox derivatives. Alternatively, [R 5 K]W 7 C was conjugated with Dox via a disulfide linker to generate [R 5 K]W 7 C-S-S-Dox conjugate, where S-S is a disulfide bond. Comparative antiproliferative assays between conjugates [R 5 K]W 7 A-Dox, [R 5 K]W 7 C-S-S-Dox, linear R 5 KW 7 A-Dox, the corresponding physical mixtures of the peptides, and Dox were performed in normal and cancer cells. [R 5 K]W 7 A-Dox conjugate was 2-fold more efficient than R 5 KW 7 A-Dox, and [R 5 K]W 7 C-S-S-Dox conjugates in inhibiting the cell proliferation of human leukemia cells (CCRF-CEM). Therefore, hybrid cyclic-linear [R 5 K]W 7 A-Dox conjugate was selected for further studies and inhibited the cell viability of CCRF-CEM (84%), ovarian adenocarcinoma (SK-OV-3, 39%), and gastric carcinoma (AGS, 73%) at a concentration of 5 μM after 72 h of incubation, which was comparable to Dox (5 μM) efficacy (CCRF-CEM (85%), SK-OV-3 (33%), and AGS (87%)). While [R 5 K]W 7 A-Dox had a significant effect on the viability of cancer cells, it exhibited minimal cytotoxicity to normal kidney (LLC-PK1, 5-7%) and heart cells (H9C2, <9%) at concentrations of 5-10 μM (compared to free Dox at 5 μM that reduced the viability of kidney and heart cells by 85% and 44%, respectively). The fluorescence microscopy images were consistent with the cytotoxicity studies, indicating minimal uptake of the cyclic-linear [R 5 K]W 7 A-Dox (5 μM) in H9C2 cells. In comparison, Dox (5 μM) showed significant uptake, reduced cell viability, and changed the morphology of the cells after 24 h. [R 5 K]W 7 A-Dox showed 16-fold and 9.5-fold higher activity against Dox-resistant cells MDA231R and MES-SA/MX2 (lethal dose for 50% cell death or LC 50 of 2.3 and 4.3 μM, respectively) compared to free Dox (LC 50 of 36-41 μM, respectively). These data, along with the results obtained from the cell viability tests, indicate comparable efficiency of [R 5 K]W 7 A-Dox to free Dox in leukemia, ovarian, and gastric cancer cells, significantly reduced toxicity in normal kidney LLC-PK1 and heart H9C2 cells, and significantly higher efficiency in Dox-resistant cells. A number of endocytosis inhibitors did not affect the cellular uptake of [R 5 K]W 7 A-Dox., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Cancer cells are known to be heterogeneous and plastic, which imparts innate and acquired abilities to resist molecular targeting by short interfering RNA (siRNA). Not all cancer cells in a population would show a similar responsiveness to targeting of genes critical for their survival and even the responders could quickly transform and switch to alternative mechanism(s) for their survival. This study was designed to look at this phenomenon by analyzing the effect of siRNA silencing of selected protein mRNAs involved in cell survival and proliferation on other protein mRNAs that could contribute to cell survival. We compared the gene expression profile of the initial population after siRNA silencing to the subpopulation that survived the siRNA silencing, to identify potential overexpressions that might explain the cell survival. Our studies show that silencing well-selected protein mRNAs simultaneously could offer advantages compared to individual siRNA silencing due to an additional impact on the expression level of certain protein mRNAs. We also demonstrate that overexpression of certain protein mRNAs could explain the innate unresponsiveness of a subpopulation of cells. These observations could be a stepping stone for further investigation of the possibility of significant synergistic effect for this combinational RNA interference strategy.

Chemotherapy is an effective approach to curb uncontrolled proliferation of malignant cells. However, most drugs rapidly lose their efficacy as a result of resistance development. We explored the potential of combinational siRNA silencing to prevent growth of drug-resistant breast cancer cells independent of chemotherapy. Resistance was induced in two breast cancer lines by chronic exposure to doxorubicin. Microarray analysis of apoptosis-related proteins showed Bcl2, survivin, NF B, and Mcl1 to be prominently up-regulated in drug-resistant cells. Human siRNA libraries against apoptosis-related proteins and kinases were screened using lipid-substituted polymers as non-viral carrier, and siRNAs were selected to diminish cell growth without affecting growth of skin fibroblasts. Surprisingly, the selected siRNAs led to similar responses in wild-type and drug-resistant cells, despite their phenotypic differences. Promising kinase siRNAs were co-delivered with anti-apoptotic Mcl-1 siRNA and Ribosomal Protein S6 Kinase (RPS6KA5) was found the most promising candidate for simultaneous silencing with Mcl-1. In both MDA435 wild type (WT) and MDA435 resistant (R) xenografts in nude mice, double silencing of Mcl-1/RPS6KA5 also led to improved inhibition of tumor growth in the absence of chemotherapy. We conclude that combinational silencing of well-selected targets could be a feasible therapeutic strategy in the absence of drug therapy and could provide a new avenue for therapy of drug-resistant breast cancers., (© 2013 Elsevier B.V. All rights reserved.)

This study aimed to develop actively targeted liposomal formulations of doxorubicin (DOX) using an engineered breast tumor targeting peptide ligand, p18-4 (WxEAAYQrFL). Towards this goal, stealth liposomes bearing different molar ratios of p18-4 peptide (1.5 and 0.3 peptide/total lipid mol %), namely HD and LD liposomes, were successfully prepared. The effect of p18-4 peptide modification and density on breast cancer cell uptake, selective cytotoxicity as well as inhibition of tumor growth and the tissue disposition of encapsulated DOX in breast tumor xenograft models in mice were assessed. The results showed a 2.4 and 5 folds decrease in the IC50 of HD liposomes in MDA-MB-435 and MCF-7 breast tumor cells, respectively. Although LD liposomes showed less (1.6 and 2.2 folds) decrease in the IC50 of DOX in the same breast cancer cell lines, they were more selective in their cytotoxic effect and uptake towards breast cancer over normal breast epithelial cells, MCF10A. Evaluation of the anticancer activity in NOD-SCID mice bearing MDA-MB-435 xenografts after receiving six i.v. injections of 2.5mg/kg/week DOX equivalent showed a superior anticancer activity for LD liposomal DOX compared to HD and unmodified liposomal formulations. Mice treated with LD liposomal DOX illustrated 4.8 folds reduction in the mean relative tumor volume compared to non-targeted DOX liposomes. This was despite similar tumor accumulation of DOX as part of LD liposomes compared to that for unmodified liposomes 24h following the last injection. In contrast, HD liposomes showed decreased DOX accumulation in the tumor and preferential uptake by liver and spleen. Treatment with unmodified and LD liposomes did not have any adverse impact on the activity level and mean body weight of live animals during the study period. In conclusion, surface modification of liposomal DOX with engineered p18-4 peptide at an optimum density can improve the antitumor efficacy and selectivity of liposomal DOX., (Copyright © 2013 Elsevier Ltd. All rights reserved.)